The present invention relates to the acid sphingomyelinase gene and to methods of diagnosing Niemann-Pick disease. It is based, at least in part, on the cloning and expression of the full-length cDNA encoding acid sphingomyelinase, the cloning and characterization of the genomic structure of the acid sphingomyelinase gene, and on the discovery of a frequent missense mutation in the acid sphingomyelinase gene of Ashkenazi Jewish Niemann-Pick disease patients.
Types A and B Niemann-Pick disease (NPD) are autosomal recessive disorders resulting from the deficient activity of the lysosomal hydrolase, acid sphingomyelinase (ASM; sphingomyelin cholinephosphohydrolase, E:C 3.1.3.12) and the accumulation of sphingomyelin, primarily in reticuloendothelial lysosomes (Niemann, 1914, Fahrb. Kinderheikd, 79:1-6; Brady et al., 1966, Proc. Natl. Acad. Sci. U.S.A. 55;366-369; Fredrickson, 1966, in xe2x80x9cThe Metabolic Basis of Inherited Disease; Stanbury et al., eds., 2nd Ed., McGraw-Hill, New York, pp. 586-602; Spence and Callahan, 1989, in xe2x80x9cThe Metabolic Basis of Inherited Disease,xe2x80x9d Scriver et al., eds., 8th Ed., McGraw-Hill, New York, pp. 1655-1676). Type A disease is a rapidly progressive neurodegenerative disease of infancy manifested by failure to thrive, severe psychomotor retardation, hepatosplenomegaly, and demise by 2-3 years of age. In comparison, type B disease is characterized primarily by reticuloendothelial system sphingomyelin deposition leading to hepatosplenomegaly and pulmonary involvement, the absence of neurologic manifestations, and survival into adulthood. The nature of the biochemical and molecular defects that underlie the remarkable clinical heterogeneity of the A and B subtypes remains unknown. Although patients with both subtypes have residual ASM activity (about 1 to 10% of normal), biochemical analysis cannot reliably distinguish the two phenotypes. Moreover, the clinical course of Type B NPD is highly variable, and it is not presently possible to correlate disease severity with the level of residual ASM activity.
Types A and B NPD occur at least 10 times more frequently among individuals of Ashkenazi Jewish ancestry than in the general population. It is estimated that the incidence of the type A disease among Ashkenazi Jews is about 1 in 40,000, a gene frequency (q) of about 1 in 200, and a heterozygote frequency (2 pq) of 1 in 100 (Goodman, 1979, in xe2x80x9cGenetic Disorders Among The Jewish Peoplexe2x80x9d, John Hopkins Univ. Press, Baltimore, pp. 96-100). The incidence of type B NPD in the Ashkenazi Jewish population is less frequent, perhaps 1 in 80 (Goodman, supra). Thus, the combined heterozygotic frequency for types A and B NPD has been estimated to be about 1 in 70 among individuals of Ashkenazi Jewish decent. Although the enzymatic diagnosis of affected patients with either type A or B NPD can be made reliably (Spence and Callahan, supra), the enzymatic detection of obligate heterozygotes has proven problematic, particularly using peripheral leukocytes as the enzyme source. Presumably, the occurrence of neutral sphingomyelinases in some sources and/or the presence of residual ASM activity resulting from the mutant allele have contributed to the inability to reliably discriminate carriers for either disease subtype. Even the use of cultured skin fibroblasts, which do not express the neutral sphingomyelinase, has not provided unambiguous results with obligate heterozygotes.
Recently, two partial cDNAs encoding human ASM were isolated and sequenced (Quintern et al., 1989, EMBO J. 8:2469-2473). The type 1 cDNA contained an in-frame 172 base pairs (bp) encoding 57 amino acids; in the type 2 cDNA this sequence was replaced by an in-frame 40 bp encoding 13 different amino acids. Of the 92 positive clones identified by cDNA library screening, the type 1 and 2 cDNAs represented about 90% and 10%, respectively (Quintern et al., supra).
The present invention relates to the ASM gene and to methods of diagnosing Niemann-Pick disease (NPD). It is based, at least in part, on the cloning and characterization of full-length cDNAs corresponding to three ASM gene transcripts and the recognition that one species of transcript could be expressed to form the active ASM enzyme. The present invention is further based on the discovery of a frequent missense mutation in the ASM gene that was detected in 32 percent of the Ashkenazi Jewish NPD type A alleles but in only 5.6 percent of ASM alleles from non-Jewish type A patients, and the discovery of a deletion mutant of the ASM gene that is associated with NPD type B disease. Additionally, the genomic sequence and structure of the ASM gene is elucidated herein.
The present invention provides for nucleic acid encoding ASM, substantially purified ASM protein and fragments and derivatives thereof, expression systems for producing ASM, genetically engineered cells and organisms containing a recombinant full-length ASM gene, probes that may be used to diagnose mutations in ASM, assay systems for the diagnosis of NPD, and methods of treatment of NPD.
In one preferred embodiment of the invention, such an assay system may be used to determine the presence of a mutation that results in an arginine to leucine substitution at amino acid residue 496 in Ashkenazi Jewish NPD patients and in prenatal diagnosis, said mutation being associated with NPD type A.
In another preferred embodiment of the invention, such an assay system may be used to determine the presence of a mutation that results in a deletion of an arginine residue at amino acid position 608 in Ashkenazi Jewish NPD patients and in prenatal diagnosis, said mutation being associated with NPD type B.